Modeled microgravity-induced protein kinase C isoform expression in human lymphocytes.

In long-term space travel, the crew is exposed to microgravity and radiation that invoke potential hazards to the immune system. T cell activation is a critical step in the immune response. Receptor-mediated signaling is inhibited in both microgravity and modeled microgravity (MMG) as reflected by diminished DNA synthesis in peripheral blood lymphocytes and their locomotion through gelled type I collagen. Direct activation of protein kinase C (PKC) bypassing cell surface events using the phorbol ester PMA rescues MMG-inhibited lymphocyte activation and locomotion, whereas the calcium ionophore ionomycin had no rescue effect. Thus calcium-independent PKC isoforms may be affected in MMG-induced locomotion inhibition and rescue. Both calcium-dependent isoforms and calcium-independent PKC isoforms were investigated to assess their expression in lymphocytes in 1 g and MMG culture. Human lymphocytes were cultured and harvested at 24, 48, 72, and 96 h, and serial samples were assessed for locomotion by using type I collagen and expression of PKC isoforms. Expression of PKC-alpha, -delta, and -epsilon was assessed by RT-PCR, flow cytometry, and immunoblotting. Results indicated that PKC isoforms delta and epsilon were downregulated by >50% at the transcriptional and translational levels in MMG-cultured lymphocytes compared with 1-g controls. Events upstream of PKC, such as phosphorylation of phospholipase Cgamma in MMG, revealed accumulation of inactive enzyme. Depressed calcium-independent PKC isoforms may be a consequence of an upstream lesion in the signal transduction pathway. The differential response among calcium-dependent and calcium-independent isoforms may actually result from MMG intrusion events earlier than PKC, but after ligand-receptor interaction.

Locomotion of lymphocytes towards melanoma cells treated with tumor necrosis factor in a syngeneic in vitro model.

Tumor necrosis factor (TNF) causes cell necrosis in vivo by damaging the endothelium of the neovasculature. However, its mechanism of action is not well understood. We hypothesized that TNF affects the tumor microenvironment even before neovascularization occurs, thereby increasing lymphocyte locomotion through the peritumoral matrix, a crucial step in tumor cell killing. The effect of TNF on lymphocytes was tested with the type I rat-tail collagen mini-assay in peripheral blood lymphocytes (PBL) from normal donors, a non-migratory PBL cell line (HPB), and a C3H mice splenic lymphocytes. Melanoma cell line (k1735p) was treated with TNFalpha/TNFbeta 10 or 20 pg/microl. The syngeneic splenic lymphocytes were layered on top of the collagen, and their migration into the collagen towards the tumor cells was assessed. Tumor cell viability was evaluated before and after TNF treatment. Paired two-tailed Student's t-test was used for statistical analysis. TNFalpha and TNFbeta had no significant direct effect on locomotion of PBL or HPB. Lymphocyte locomotion was inhibited in the presence of untreated melanoma cells in 7 of 9 assays (statistically significant in four), and it was significantly increased towards TNFalpha- or beta-treated melanoma cells, compared to untreated condition, in 7 of 9 assays (p=0.05 to p=0.0001). The number of viable tumor cells was not significantly different before and after treatment. In conclusion, treatment of tumor cells with TNFalpha or TNFbeta significantly enhances lymphocyte locomotion through the matrix. The effect of TNF is not the result of a direct influence on the lymphocytes, and is not associated with a decrease in the number of viable tumor cells. These findings suggest that TNF interaction with the cell microenvironment induces a change in lymphocyte locomotion.

Suppression of antigen-specific lymphocyte activation in modeled microgravity.

Various parameters of immune suppression are observed in lymphocytes from astronauts during and after a space flight. It is difficult to ascribe this suppression to microgravity effects on immune cells in crew specimens, due to the complex physiological response to space flight and the resultant effect on in vitro immune performance. Use of isolated immune cells in true and modeled microgravity in immune performance tests, suggests a direct effect of microgravity on in vitro cellular function. Specifically, polyclonal activation of T-cells is severely suppressed in true and modeled microgravity. These recent findings suggest a potential suppression of oligoclonal antigen-specific lymphocyte activation in microgravity. We utilized rotating wall vessel (RWV) bioreactors as an analog of microgravity for cell cultures to analyze three models of antigen-specific activation. A mixed-lymphocyte reaction, as a model for a primary immune response, a tetanus toxoid response and a Borrelia burgdorferi response, as models of a secondary immune response, were all suppressed in the RWV bioreactor. Our findings confirm that the suppression of activation observed with polyclonal models also encompasses oligoclonal antigen-specific activation.

Modeled microgravity inhibits apoptosis in peripheral blood lymphocytes.

Microgravity interferes with numerous lymphocyte functions (expression of cell surface molecules, locomotion, polyclonal and antigen-specific activation, and the protein kinase C activity in signal transduction). The latter suggests that gravity may also affect programmed cell death (PCD) in lymphocyte populations. To test this hypothesis, we investigated spontaneous, activation- and radiation-induced PCD in peripheral blood mononuclear cells exposed to modeled microgravity (MMG) using a rotating cell culture system. The results showed significant inhibition of radiation- and activation-induced apoptosis in MMG and provide insights into the potential mechanisms of this phenomenon.

Microarray analysis of genes differentially expressed in HepG2 cells cultured in simulated microgravity: preliminary report.

Developed at NASA, the rotary cell culture system (RCCS) allows the creation of unique microgravity environment of low shear force, high-mass transfer, and enables three-dimensional (3D) cell culture of dissimilar cell types. Recently we demonstrated that a simulated microgravity is conducive for maintaining long-term cultures of functional hepatocytes and promote 3D cell assembly. Using deoxyribonucleic acid (DNA) microarray technology, it is now possible to measure the levels of thousands of different messenger ribonucleic acids (mRNAs) in a single hybridization step. This technique is particularly powerful for comparing gene expression in the same tissue under different environmental conditions. The aim of this research was to analyze gene expression of hepatoblastoma cell line (HepG2) during early stage of 3D-cell assembly in simulated microgravity. For this, mRNA from HepG2 cultured in the RCCS was analyzed by deoxyribonucleic acid microarray. Analyses of HepG2 mRNA by using 6K glass DNA microarray revealed changes in expression of 95 genes (overexpression of 85 genes and downregulation of 10 genes). Our preliminary results indicated that simulated microgravity modifies the expression of several genes and that microarray technology may provide new understanding of the fundamental biological questions of how gravity affects the development and function of individual cells.

Induction of three-dimensional assembly of human liver cells by simulated microgravity.

The establishment of long-term cultures of functional primary human liver cells (PHLC) is formidable. Developed at NASA, the Rotary Cell Culture System (RCCS) allows the creation of the unique microgravity environment of low shear force, high-mass transfer, and 3-dimensional cell culture of dissimilar cell types. The aim of our study was to establish long-term hepatocyte cultures in simulated microgravity. PHLC were harvested from human livers by collagenase perfusion and were cultured in RCCS. PHLC aggregates were readily formed and increased up to 1 cm long. The expansion of PHLC in bioreactors was further evaluated with microcarriers and biodegradable scaffolds. While microcarriers were not conducive to formation of spheroids, PHLC cultured with biodegradable scaffolds formed aggregates up to 3 cm long. Analyses of PHLC spheroids revealed tissue-like structures composed of hepatocytes, biliary epithelial cells, and/or progenitor liver cells that were arranged as bile duct-like structures along nascent vascular sprouts. Electron microscopy revealed groups of cohesive hepatocytes surrounded by complex stromal structures and reticulin fibers, bile canaliculi with multiple microvilli, and tight cellular junctions. Albumin mRNA was expressed throughout the 60-d culture. A simulated microgravity environment is conducive to maintaining long-term cultures of functional hepatocytes. This model system will assist in developing improved protocols for autologous hepatocyte transplantation, gene therapy, and liver assist devices, and facilitate studies of liver regeneration and cell-to-cell interactions that occur in vivo.

Cultures of human liver cells in simulated microgravity environment.

We used microgravity-simulated bioreactors that create the unique environment of low shear force and high-mass transfer to establish long-term cultures of primary human liver cells (HLC). To assess the feasibility of establishing HLC cultures, human liver cells obtained either from cells dissociated by collagenase perfusion or minced tissues were cultured in rotating vessels. Formation of multidimensional tissue-like spheroids (up to 1.0 cm) comprised of hepatocytes and biliary epithelial cells that arranged as bile duct-like structures along newly formed vascular sprouts were observed. Electron microscopy revealed clusters of round hepatocytes and bile canaliculi with multiple microvilli and tight junctions. Scanning EM revealed rounded hepatocytes that were organized in tight clusters surrounded by a complex mesh of extracellular matrix. Also, we observed that co-culture of hepatocytes with endothelial cells stimulate albumin mRNA expression. In summary, a simulated microgravity environment is conducive for the establishment of long-term HLC cultures and allows the dissection of the mechanism of liver regeneration and cell-to-cell interactions that resembles in vivo conditions.

Changes in gravity inhibit lymphocyte locomotion through type I collagen.

Immunity relies on the circulation of lymphocytes through many different tissues including blood vessels, lymphatic channels, and lymphoid organs. The ability of lymphocytes to traverse the interstitium in both nonlymphoid and lymphoid tissues can be determined in vitro by assaying their capacity to locomote through Type I collagen. In an attempt to characterize potential causes of microgravity-induced immunosuppression, we investigated the effects of simulated microgravity on human lymphocyte function in vitro using a specialized rotating-wall vessel culture system developed at the Johnson Space Center. This very low shear culture system randomizes gravitational vectors and provides an in vitro approximation of microgravity. In the randomized gravity of the rotating-wall vessel culture system, peripheral blood lymphocytes did not locomote through Type I collagen, whereas static cultures supported normal movement. Although cells remained viable during the entire culture period, peripheral blood lymphocytes transferred to unit gravity (static culture) after 6 h in the rotating-wall vessel culture system were slow to recover and locomote into collagen matrix. After 72 h in the rotating-wall vessel culture system and an additional 72 h in static culture, peripheral blood lymphocytes did not recover their ability to locomote. Loss of locomotory activity in rotating-wall vessel cultures appears to be related to changes in the activation state of the lymphocytes and the expression of adhesion molecules. Culture in the rotating-wall vessel system blunted the ability of peripheral blood lymphocytes to respond to polyclonal activation with phytohemagglutinin. Locomotory response remained intact when peripheral blood lymphocytes were activated by anti-CD3 antibody and interleukin-2 prior to introduction into the rotating-wall vessel culture system. Thus, in addition to the systemic stress factors that may affect immunity, isolated lymphocytes respond to gravitational changes by ceasing locomotion through model interstitium. These in vitro investigations suggest that microgravity induces non-stress-related changes in cell function that may be critical to immunity. Preliminary analysis of locomotion in true microgravity revealed a substantial inhibition of cellular movement in Type I collagen. Thus, the rotating-wall vessel culture system provides a model for analyzing the microgravity-induced inhibition of lymphocyte locomotion and the investigation of the mechanisms related to lymphocyte movement.

Impairment of lymphocyte locomotion in the tumor microenvironment and the effect of systemic immunotherapy with liposome-encapsulated muramyl-tripeptide-phosphatidylethanolamine.

The ability of the lymphocytes to move through the interstitium is obligatory to the immune response. We previously showed that tumor-infiltrating lymphocytes (TIL) from human melanoma and renal cell carcinoma demonstrate a dramatic decrease in their spontaneous locomotion through three-dimensional collagen gel when compared with peripheral blood lymphocytes (PBL) and lymph node lymphocytes. To determine if this decrease is caused by contact with tumor cells, or mediated through certain diffusible factors, we examined the effects of autologous tumor cells on the locomotion of PBL in a model system where tumor cells were separated from lymphocytes by a 3-mm layer of gelled collagen. After 21-22 h incubation in chamber slides, locomotion distances were assessed in the presence and absence of tumor and normal cells. In the presence of tumor cells, PBL from 14 of 18 patients displayed substantial (466.5 +/- 2.7 microns compared to control 568.9 +/- 10.9 microns, P < 0.001) loss of motility. Inhibition was more prominent in melanoma patients than in renal cell carcinoma patients. Thus the impaired locomotion previously observed in TIL was at least partially due to the presence of tumor. The locomotion of TIL was restored in four of five melanoma patients treated with liposome-encapsulated muramyl-tripeptide-phosphatidylethanolamine (L-MTP-PE). Furthermore, in six of seven examined L-MTP-PE-treated patients, an increase in intrinsic PBL locomotion during the first month of the therapy was observed. These results suggest that the environment of the tumor is not conducive to locomotion of advancing lymphocytes and the therapeutic intervention may ameliorate the loss of lymphocytic infiltration.

Effect of surgery on peripheral blood lymphocyte locomotion through type I collagen.

BACKGROUND: Locomotion of peripheral blood lymphocytes (PBL) through peritumoral matrix is obligatory for tumor cell killing. The authors investigated the effect of surgery on lymphocyte locomotion and compared it with the effect on natural killer cell cytotoxicity (NKCC). METHODS: PBL from 12 patients with cancer were assessed for locomotion in Type I rat-tail collagen. Preoperative and postoperative locomotion (after 20 hours of incubation) and NKCC were estimated. RESULTS: Locomotion of lymphocytes through collagen increased significantly after operation in 6 of 12 patients, whereas only 1 of 12 had a decrease (P < 0.001). Short-term (20-hour incubation) exposure of the locomotory HSB cell line to patient plasma samples did not affect their migration. NKCC, as estimated against K562 target cells with the use of the 51Cr-release assay, decreased 5-50% after operation in 9 of 12 patients (P = 0.006). No correlation could be demonstrated between the changes in locomotion and NKCC (regression analyses), nor were identifiable clinical factors associated with these changes. CONCLUSIONS: Locomotion of PBL through collagen increases after operation in patients with cancer, whereas possibly independent factors may decrease postoperative NKCC.

Locomotion through three-dimensional type I rat tail collagen. A modified mini-assay.

There is an increased application of three-dimensional type I rat tail collagen as an in vitro model for the peritumoral matrix in analysis of lymphocyte migration. The increased demand prompted us to modify the previous methods. We here describe our 'mini'-setup of the collagen model assay, which uses only 1/20 the amount of collagen medium and the number of cells used in the conventional assay. The modified assay was tested for optimal collagen concentration in gel for upward and downward migration, for locomotion from a collagen-gel bead into a collagen overlayer for demonstration of the effect of inhibitors and for differentiation between locomotory properties of lymphocyte subpopulations. The results verify that the mini-assay is an applicable in vitro model, easily read and amenable to limited blood samples such as those obtained from cancer patients, and reflects well known in vivo events.